Tuberous Sclerosis (TSC) is caused by mutation in either TSC1 or TSC2 gene and is characterized by the formation of hamartomas in a variety of organs. However, the development of malignancy is very rare in TSC. TSC1-TSC2 complex is a critical negative regulator of the mammalian target of rapamycin (mTOR). It has been showed that an mTOR-mediated negative feedback loop leads to attenuation of Akt signaling in TSC-related tumors and compromised cell survival in TSC deficient cells, which might explain the rare progression to malignancy in TSC. However, uncertainties remain as to (i) the underlying mechanisms and key downstream effector(s) of Akt that mediate the pro-survival function of TSC and (ii) the basis for the attenuated cancer phenotype of the TSC-related tumors. Recent genetic studies from both mouse models and human cancers strongly suggest that the FoxO transcription factors represent key downstream effector arms of PI3K-AKT signaling network and play critical roles in cancer development. Indeed, neoplastic conversion of endothelial cells is a prominent phenotype upon loss of either TSC or FoxO, suggesting an epistatic relationship. Furthermore, FoxOs were shown to localize in the nucleus and active in TSC deficient cells and tumors, suggesting that FoxOs might play a critical role in TSC pathophysiology. Thus, a clear understanding the genetic and functional interactions between TSC and FoxO appears to be critical to understand TSC pathogenesis and ultimate management. This proposal aims to elucidate the underlying mechanisms of rare malignancy in TSC-related tumors. We hypothesize that the anti-apoptotic function of TSC is mainly mediated by suppression of FoxOs transcriptional activity through promoting Akt-mediated FoxO phosphorylation. Reactivation of FoxOs upon loss of TSC might contribute to the less severity of TSC-related tumors and thus compound deletion of both TSC and FoxOs would lead more malignant phenotypes. The specific aims are (1) to examine whether compound inactivation of FoxOs and TSC in the mouse would exacerbate the tumor phenotypes observed in TSC single mutants, (2) to study the impact of FoxOs on TSC-mediated cell biological activity in primary cells, and (3) to identify direct FoxO transcriptional targets which might mediate the cell biological functions of TSC. This proposal will provide important biochemical and biological insights into the role and essentiality of FoxO transcriptional factors in TSC development. The negative feedback regulation of Akt signaling by TSC-mTOR signaling suggests combined treatment of rapamycin analogues with inhibitors of PI3K-Akt signaling should be used in TSC clinical trials. Given the critical role of FoxO in PI3K-Akt signaling, our efforts to identify direct FoxO transcriptional targets involved in the regulation of TSC might expand drug development opportunities for TSC or, minimally, identify a FoxO biomarker which may provide a pharmacodynamic marker for monitoring the impact of anti-PI3K drugs in clinical trials. PUBLIC HEALTH RELEVANCE: This proposal aims to examine the role of FoxO transcriptional factors, a key downstream surrogate of PI3K-AKT signaling in cancer development, in TSC-mediated negative feedback regulation through combined genetic, genomics and cell biological approaches. The identification of validated FoxO targets with oncogenic activities playing essential roles in the regulation of TSC may expand drug target opportunities for this disease. 1